Identification and characterization of salt-tolerance relative miRNAs in Procambarus clarkii by high-throughput sequencing
- 55 Downloads
Procambarus clarkii is one of the important economic species in China and has been served as tasty food in recent years after being introduced to Nanjing. Significant problems of environment factors, such as salinity, pH and temperature, especially salinity, has the potential to result in significant economic losses in many crayfish-producing farms in China. miRNAs are a kind of ~ 22 nucleotide small non coding RNAs which were encoded by plants, animals and some viruses with functions in RNA silencing or post-transcription regulation. We constructed four sRNA library of P. clarkia from different tissues and treatments by using high-throughput sequencing technology. A total of 101 conserved miRNAs and two novel pre-miRNAs were identified and RT-qPCR were further performed to confirm existence of part of identified miRNAs. A genome wide expression profile of salt-tolerance miRNAs was proved and three miRNAs were further validated by RT-qPCR with dynamic response to different salinity stages. The study of miRNAs in P. clarkia can help us better understanding the role of miRNAs in salt-tolerance in P. clarkia.
KeywordsmiRNA Crayfish Procambarus clarkia High-throughput sequencing Salt tolerance
Procambarus clarkii is the most cosmopolitan crayfish around the world. In some countries, P. clarkii is a species of great commercial interest . This crayfish is one of the important economic species in China and has been served as tasty food in recent years after being introduced to Nanjing, China from Japan in 1929 . P. clarkia can tolerate extreme and polluted environments and served as an indicator of metal pollution in numerous studies of aquatic environments . Generally, P. clarkia has great resistance to diseases in natural environments. Nevertheless, the present sustainability and healthy development of the crayfish aquaculture are at risk due to significant problems of environment factors, such as salinity, pH and temperature. These factors, especially salinity, has the potential to result in significant economic losses in many crayfish-producing farms in China. Under these circumstances, an investigation into the mechanisms of salt-tolerance of P. clarkii might be beneficial to the management of crayfish farming.
MicroRNAs (miRNAs) are a kind of ~ 22 nucleotide small non coding RNAs which were encoded by plants, animals and some viruses [4, 5, 6]. miRNAs have great functions in RNA silencing or post-transcription regulation via base-pairing with complementary sequences in mRNAs . miRNAs are abundant in many cell types [8, 9] and can regulate nearly 60% of genes in mammals [10, 11]. A total of 10,000 different miRNAs had been identified and reported in miRBase for all species . Expression profile of miRNAs changed a lot in pathological state or environmental factor stimulation. Previous studies have reported the miRNA profiles in P. clarkia, for example, Wang et al. and Du et al. identified the miRNAs in gills, intestines and lymph organs of P. clarkia infected with white spot syndrome virus [13, 14, 15]. Ou et al. screened the miRNAs potentially related to immunity against Spiroplasma eriocheiris infection in P. clarkia . However, the miRNA profiles in P. clarkia under environmental factor stimulation have never been reported. This study of miRNAs in P. clarkia can help us better understanding the role of miRNAs in salt-tolerance in P. clarkia.
In this study, we used high-throughput sequencing technology and bioinformatics analysis to identify conserved and novel miRNAs in P. clarkii. In addition, the possible salt-tolerance relative miRNAs of P. clarkii were analyzed.
High-throughput sequencing of P. clarkia small RNAs
Identification of conserved miRNAs in P. clarkia
Identification of novel miRNAs in P. clarkia
Analysis of miRNA involved in salt tolerance in P. clarkia
In this study, we used high-throughput sequencing technology to identify potential miRNAs of P. clarkia. We used SOAP (http://soap.genomics.org.cn) [19, 20] to align sRNA reads in our libraries to known miRNA in miRBase v19. If we can find perfect match sequences of one miRNAs in our libraries, certain miRNAs were thought to be existed in P. clarkia. However, whole genome of P. clarkia was not reported yet, it was hard to prove that the sequences we detected were from P. clarkia or contaminations. Although we used semi-quantitative RT-PCR and RT-qPCR to further prove the reliability of part identified miRNAs. More experiments should be done to prove the existence of miRNAs in P. clarkia. Two novel pre-miRNAs were also identified using ESTs in this study. ESTs can’t reflect whole genome of P. clarkia, more novel pre-miRNAs could be found if we got whole genome of P. clarkia.
Among 9 miRNAs detected by RT-qPCR assasys, miR-276, let-7a-5p and miR-71 showed negative correlation with salt-tolerance. For transcriptome of P. clarkia was not sequenced yet, we cannot search possible target genes of these miRNAs as we usually do. Previous study reported that Na+-K+-ATPase was mainly located in crustacean gill and its activities had positive correlation with salinity level [21, 22]. The identified salt-tolerance relative miRNAs may play roles in Na+-K+-ATPase pathways. Further researches can be deployed to study the relationship between miRNA and Na+-K+-ATPase activities.
We have proven that salinity level has effects on miRNA profile of P. clarkia in this study. Other factors can also have effects on miRNA profile of P. clarkia. Emodin diet and white spot syndrome virus have been proven that can influence miRNA abundances of P. clarkia and miRNAs played important roles in immunity, RNA transport and other important biological progresses [15, 23]. The study of miRNAs in P. clarkia contributes a better understanding of miRNA function in crayfish.
We constructed four sRNA library of P. clarkia from different tissues and treatments by using high-throughput sequencing technology. A total of 101 conserved miRNAs and two novel pre-miRNAs were identified and RT-qPCR were further performed to confirm existence of part of identified miRNAs. A genome wide expression profile of salt-tolerance miRNAs was proved and three miRNAs were further validated by RT-qPCR with dynamic response to different salinity stages. High-throughput sequencing provides an opportunity to analyze salt-tolerance relative miRNAs in P. clarkia, which will help to unravel new components of salt stress pathways and gain new insights into gene function and regulation in crayfish.
Experimental animal collection and RNA isolation
All sampels of P. clarkia were bought from local market. Total RNA was isolated using TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions.
The high-throughput sequencing was conducted by using Illumina Genome Analyzer IIx according to the manufacture’s protocols. Small RNA molecules (18-30 nt) were purified from total RNA using PAGE gel and were then used for library preparation according Illumina TruSeq Small RNA Sample Preparation Guide. Briefly, after ligation of 3′ and 5′ adapters to their both ends, the RNA samples were then amplified by using adapter primers for 17 cycles. The PCR products (around 147 bp) were isolated from agarose gels and directly used for cluster generation. Small RNA library was then sequenced using Illumina Genome Analyzer IIx. CASAVA 1.5 was used to get raw sequencing data from the image files generated by the machines. Quality control of raw sequencing data was performed by fastx-toolkit. After filtering the low-quality reads and trimming 3′ adaptor sequencing and removing 5′ adaptor and polyA contaminations, clean reads were processed for following analysis.
In silico analysis
To identify known miRNAs P. clarkii, high-throughput sequencing reads were aligned against all known miRNA precursors and mature miRNAs present in miRBase database with SOAP [19, 20]. Sequences not matched in above databases were remained for further analysis.
To identify novel miRNAs in P. clarkii, EST sequences of P. clarkia were collected from GenBank database in NCBI. SOAP was also used to align remained sequences to ESTs. MIREAP is used to identify genuine miRNAs from 4 constructed small RNA libraries combining miRNA biogenesis, sequencing depth and structural features. All pre-miRNA candidates were subjected to MiPred to filter out pseudo-pre-miRNAs. True pre-miRNA candidates were used for further analysis.
Quantitative RT-PCR assays
Quantitative RT-PCR was performed using TaqMan miRNA probes (Applied Biosystems, Froster City, CA, USA) using an ABI-7300 PCR machine according to the manufacturer’s instructions. The amount of RNA input was o.5μg. Relative expression of miRNAs in tissues was determined after normalization to β-actin mRNA levels.
This work was supported by grants from Nanjing University Innovation and Creative Program for PHD candidate (No. 2016025).
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
FFJ conceived and designed the experiments. YLS and FFJ participated in the bioinformatic analysis, experiments and drafted the manuscript. Both authors read and approved the final manuscript.
Ethics approval and consent to participate
Consent for publication
We have obtained consents to publish this paper from all the participants of this study.
The authors declare that they have no conflict of interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- 12.S. Griffiths-Jones, miRBase: microRNA sequences and annotation, Curr Protoc Bioinformatics, Chapter 12 (2010) Unit 12 19 11–10.Google Scholar
- 14.Du Z-q, Wang K, Shen X-l, Jin Y-h, Jin H-x, Li X-c. Identification and characterization of intestine microRNAs and targets in red swamp crayfish, Procambarus clarkii infected with white spot syndrome virus. PLoS One. 2017;12(11).Google Scholar
- 16.Ou J, Li Y, Ding Z, Xiu Y, Wu T, Du J, Li W, Zhu H, Ren Q, Gu W, Wang W. Transcriptome-wide identification and characterization of the Procambarus clarkii microRNAs potentially related to immunity against Spiroplasma eriocheiris infection. Fish Shellfish Immunol. 2013;35:607–17.CrossRefGoogle Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.